A number of miRNAs have been identified to be involved in the process of in- stent restenosis and therefore modulating their expression could lead to a miRNA-based therapeutic to inhibit neointimal growth. A summary of these miRNAs is given in (Table 1).
References miRNA After injury Target
Cell Molecular Targets (Ji et al., 2007b, McDonald et al., 2015)
miR-21 upregulated VSMC PTEN, PDCD4
(Cheng et al., 2009b)
miR-145 downregulated VSMC ACE, KLF5
(Cordes et al., 2009b)
miR-143 downregulated VSMC ELK1, FRA1
(Davis et al., 2009) miR-221 upregulation VSMC p27, C-KIT (Iaconetti et al.,
2012)
miR-92a upregulation EC KLF4, MKK4
Table 1 miRNAs associated with neointima formation which could be targets for treatment of ISR.
1.6.2.1 miRNA-21
miRNA-21 has been identified as a potential miRNA which could be used to modulate the wound healing process inflicted from stent deployment. A microarray was conducted on rat arteries before and after balloon injury, which indicated several miRNAs were aberrantly expressed before and after injury. miRNA-21 was one of the miRNAs which exhibited a dramatic change in expression resulting in a 5 fold increase at 7 days after injury. Therefore, knockdown of this miRNA was hypothesised as a strategy to inhibit neointima formation using antisense-miRNA-21. In vitro experiments confirmed a dose dependent response on antisense-miRNA-21 with decreased apoptosis and cell
proliferation, which was conformed in vivo using a ratballoon injury model, indicating that treatment of the vessel with antisense-miRNA-21 inhibited neointima formation (Ji et al., 2007b).
miRNA-21 has also been shown to be dysregulated after porcine vein grafting at 7 and 21 days, and has been identified as having a functional role in neointimal growth within this setting (McDonald et al., 2013). Using miRNA-21 knock-out (KO) mice, miRNA-21s role in neointima formation after vein graft surgery was investigated, indicating that the absence of miRNA-21 had positive effects on preventing neointima formation. Due to the similar pathologies between neointima formation in vein graft surgery and in-stent restenosis, it was likely that miRNA-21 may play a similar role in vessels which have been injured through stenting.
In an in-vivo porcine study, both lead and passenger strands were shown to be upregulated 7 and 28 days after stenting, suggesting that this miRNA might play an important role in the post-stent injury response. A further investigation was conducted using miRNA-21 (KO) mice, which revealed that the neointima area, neointimal thickness and neointima/medial ratio was drastically decreased when compared to the WT controls, 28 days after stenting, giving support to the hypothesis that miRNA-21 could play a pivotal role in neointima formation. In order to investigate potential mechanisms which could help explain these observations, proliferation and migration assays were conducted with platelet derived growth factor (PDGF) as a stimulant on cultured miR-21 KO SMCs against wild type (WT) control cells. The miR-21 KO SMC showed attenuated proliferation and migration responses to this stimulus when compared to the KO control SMCs (McDonald et al., 2015). These results suggest that downregulating miR-21 expression locally to stented vessels could provide a therapy which could supress neointima formation and ISR.
1.6.2.2 miR-145 and miR-143
miR-145 is the most abundantly expressed miRNA in normal vascular walls and VSMCs which have been freshly isolated. However, expression of miR-145 is almost undetectable in ECs (Ji et al., 2007b, Cheng et al., 2009b). An interesting observation was made that the expression of miR-145 was supressed in vessel
walls with neointimal growth (Ji et al., 2007b), leading to the hypothesis that miR-145 may have a regulatory role in modulating the SMC phenotype.
This theory was tested by monitoring the expression of miR-145 in PDGF stimulated VSMCs. PDGF is well-known to promote SMC proliferation and migration, by promoting a change in the SMC phenotype from contractile to synthetic states; this can be monitored by dedifferentiation markers such as SM α-actin, calponin and SM-MHC. It was observed that treatment of the cells with PDGF caused a decrease in mir-145 levels. Furthermore, overexpression of miR- 145 was found to inhibit PDGF-induced VSMC dedifferentiation. Conversely, inhibition of miR-145 expression resulted in increasing levels of PDGF-induced VSMC dedifferentiation (Cheng et al., 2009b).
The role of miR-145 on phenotype modulation of VSMCs was then investigated in the absence of PDGF stimulation, with VSMCs in the contractile state and interestingly it was shown that overexpression and inhibition of miR-145 levels resulted in decrease and increase in the synthetic phenotype VSMC markers respectively (Cheng et al., 2009b). Inhibition of miR-145 immediately after stenting could therefore reduce neointima formation, by sequestering the VSMC response to injury and phenotype switching through the modulation of the miR- 145 expression.
In the same year, other key findings were published with regards to the miR143/145 gene cluster role in SMC differentiation. Cordes et al. reported that myocardin upregulated miR-143 and 145 expression culminating in the induction of SMC marker gene expression and myofilament formation (Cordes et al., 2009b). Boettger et al. demonstrated that, VSMCs from a miR-143/145 KO mouse exhibited VSMCs which did not express calponin, smoothelin, which are key contractile VSMC markers. The structure of the arteries also lacked myofilamentous cytoskeletal organisation, suggesting the key role that miR- 143/145 gene cluster plays in the development of the vasculature and again highlighting its potential use as a therapeutic to regain vessel homeostasis after injury (Boettger et al., 2009).
1.6.2.3 miR-221
miR-221 has also been implicated as a potential key regulator of VSMC phenotype, through action via the PDGF signalling pathway. PDGF is well known to promote neointima formation inducing SMC phenotype switching. In a study directed by Davis et al.,miR-221 was found to be transcriptionally induced by treatment of the VSMCs with PDGF, leading to downregulation of p27Kip1. The action of miR-221 on p27Kip1 was shown to be essential for PDGF induced VSMC proliferation (Davis et al., 2009). These findings provide another example of how miRNAs play an important role in the maintenance of SMC behaviour, which leads to the development of them being used as potential therapeutics within the setting of stent deployment. In this case, if a miR-221 inhibitor was administered immediately after stenting, this could act as a suppressor for the PDGF pathway.
1.6.2.4 miR-92a
Tackling ISR by promoting re-endothelialisation could be achieved by the administration of a miR-92a inhibitor. This has been shown to be highly expressed in ECs but not in VSMCs and has been identified to promote EC proliferation when downregulated in vitro, using a BrdU assay. Delivery of antago-miR-92a has also been shown to be effective at preventing neointima formation in vivo after balloon injury of rat carotid arteries. The mode of action that miR-92a was having on EC function was investigated and it was found that key EC intracellular signalling molecules, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) were increasingly phosphorylated upon inhibition of miR-92a. In addition, through loss of function assays, mitogen- activated protein kinase kinase 4 (MKK4) and Kruppel-like factor 4 (KLF4) were found to be mRNA targets for miR-92a (Iaconetti et al., 2012).